ReproTracker®

ReproTracker® is a state-of-the-art in vitro assay to visualize the key events during early embryonic development that allows us to reliably assess the potential developmental toxicity hazards of new drugs and chemicals. This unique assay provides insight into the toxic mode-of-action.

Key Features

        • Biomarker assay for developmental toxicity
        • Visualisation of key developmental pathways
        • Insight into the mode-of-action
        • Human stem cell-based assay

Stem cells are widely accepted as an excellent platform for in vitro developmental toxicity hazard identification. ReproTracker® is a human induced pluripotent stem cell (hiPSC) assay, which uses a specific set of biomarkers to monitor the various stages of early embryonic development. The ReproTracker biomarkers represent specific phases of differentiation from pluripotent stem cell into different mature tissues. When the differentiating stem cells progress through the different stages of differentiation, expression of the selected biomarker genes will be activated or deactivated.

Disruption of proper stem cell differentiation following chemical exposure indicates their potential teratogenic properties. During stem cell differentiation, the developing tissues are continuously monitored for adverse effects and morphological abnormalities compared to unexposed cultures. Furthermore, activation of various lineage-specific biomarkers during differentiation is determined to investigate the mechanism of developmental toxicity.

In ReproTracker, the hiPS cells are differentiated in to functional hepatocytes and cardiomyocytes.

 

Hepatocyte Differentiation

Overview of hiPSC differentiation protocols towards hepatocytes. hiPSC were differentiated over 21 days by the addition of specific growth factors. Effective differentiation was confirmed by measuring reference gene expression changes using qPCR and immunostaining of Albumin

 

Cardiomyocyte differentiation

Overview of hiPSC differentiation protocols towards cardiomyocytes. hiPSC were differentiated over 14 days by the addition of specific growth factors. Effective differentiation was confirmed by measuring reference gene expression changes using qPCR and immunostaining of Troponin T2 (TNNT2).

ReproTracker Validation

ReproTracker has been validated for the detection of in vivo teratogenicity using a compound set of 39 teratogens and 14 non-teratogens. hiPSC cells were exposed during differentiation to functional cardiomyocytes and hepatocytes. Tissue morhphology, toxicity and biomarker expression changes were analysed at various time points during differentiation.

Prediction in the ReproTracker assay is compared with the in vivo classification of the tested compounds. The compounds were classified in ReproTracker as in vivo teratogens or non-teratogens based on expert judgement.  Validation of the assay indicated a 79% predictability, 87% sensitivity and  57% specificity for the identification of teratogenicity.

Results for teratogenic agents

Valproic acid was tested at 4 non-cytotoxic and 1 cytotoxic concentrations. Changes in the morphology of both differentiated hepatocytes and cardiomyocytes were observed at non-cytotoxic concentrations. The marked grey area (*) represents the data that is excluded from the analysis due to the significant increase of observed cytotoxicity at the final stage of differentiation. Expression of both AFP and MYH6 in the differentiated hepatocytes and cardiomyocytes was below the set threshold for developmental toxicity. Therefore, valproic acid was classified as teratogen in the ReproTracker, which is in agreement with its in vivo classification. 

 

The nucleoside analogue Hydroxyurea was tested at 4 non-cytotoxic and 1 cytotoxic concentrations. Changes in the morphology of both differentiated hepatocytes and cardiomyocytes were observed at non-cytotoxic concentrations. The marked grey area (*) represents the data that is excluded from the analysis due to the significant increase of observed cytotoxicity at the final stage of differentiation. Expression of both AFP and MYH6 in the differentiated hepatocytes and cardiomyocytes was below the set threshold for developmental toxicity. Therefore, hydroxyurea was classified as teratogen in the ReproTracker, which is in agreement with its in vivo classification. 

 

Results for none-teratogenic compounds

The antibiotic Amoxicillin was tested at 5 non-cytotoxic concentrations. Changes in the morphology of both differentiated hepatocytes and cardiomyocytes were not observed at any of the tested  concentrations. Expression of both AFP and MYH6 in the differentiated hepatocytes and cardiomyocytes was above the set threshold for developmental toxicity. Therefore, amoxicillin was classified as non-teratogen in the ReproTracker, which is in agreement with its in vivo classification.

 

The channel modulator Chlorthalidone was tested at 5 non-cytotoxic concentrations. Changes in the morphology of both differentiated hepatocytes and cardiomyocytes was not observed at any of the tested concentrations. The marked grey area (*) represents the data that is excluded from the analysis due to the significant increase of observed cytotoxicity at the final stage of differentiation. Expression of both AFP and MYH6 in the differentiated hepatocytes and cardiomyocytes was above the set threshold for developmental toxicity. Therefore, chlorthalidone was classified as non-teratogen in the ReproTracker, which is in agreement with its in vivo classification. 

From sample to report

Reception of your test compound at the Toxys lab is where our ReproTracker service starts.  Below is a visualisation of the sequence of events between receiving your test compound,  performing the ReproTracker assay and discussing the final results.

ReproTracker experimental design

ReproTracker services

Toxys offers ReproTracker as as a service from our state-of-the-art laboratory in Leiden, the Netherlands. You can then send your compounds and receive a full report, in most cases within 9-12 weeks.

 

Compound requirement

  • 50 mg test material requested
  • Top concentration tested in ReproTracker is  1 mM or 1 mg/ml

 

Turn around time

  • 9-12 weeks 

 

Throughput

  • 4-8 compounds per week 

 

Type of solvents

  • DMSO/PBS
  • Water

 

Protocol

  • Extensive cytotoxicity dose range finding, twenty concentrations
  • Differentiation of ReproTracker cells into liver and heart
  • Five compound concentrations in ReproTracker
  • Morpohological profiling, cytotoxicity assessment and biomaker analysis by quantitative real-time PCR
  • Positive and negative controls included in every test
  • Three technical repeats
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